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Category Archives: Transfection

Plasmid DNA and siRNA transfection protocol optimization

There are several experimental parameters that affect transfection efficiency and associated cell viability for siRNA and plasmid DNA transfection experiments:

  • Transfection protocol (method and conditions)
  • Health of cultured cells
  • Purity and concentration of pDNA and/or siRNA
  • Controls

Transfection protocol

Optimized transfection protocols are available for over 100 cancer cell lines and primary cell types (see Transfection Kits for Cell Lines). For in vivo delivery there are lipid based, polymer based, nanoparticle based, and PEG liposome based transfection reagents with optimized transfection protocols are available (see In Vivo Transfection Reagents), as well as tissue-targeted delivery reagents (see Kidney, Liver, Pancreas tissue targeted transfection kits).

Health of cultured cells

We do not recommend using freshly thawed cells for transfection experiments. Cells should be passaged 2-3 times and exhibit exponential growth at the time of transfection experiment. Use of antibiotics in cultured cell medium can interfere with transfection and reduce both transfection efficiency and cell viability of transfected cells. Cells and media must be free of contamination (mycoplasma, bacteria, yeast).

Cargo molecule: pDNA or siRNA

Purity and lack of degradation is very important for successful transfection experiment. Dilutions of pDNA and siRNA solutions should be done using RNase- and DNase-free water (or TE-buffer). Incomplete or low grade purification can lead to presence of such contaminants as endotoxins, proteins, lipids, carbohydrates that may induce siRNA and pDNA degradation and lack of functional performance (pDNA expression and siRNA gene silencing efficacy). Scientific Calculators can help with experimental planning, nucleic acid amount and concentration calculation, and associated conversions.

Controls

Positive and negative transfection controls are crucial for transfection experiments and associated data analysis. Commercially available siRNA and pDNA controls available from Altogen Biosystems:

  • Catalog #4060 – GFP-expressing plasmid DNA (25 ug)
  • Catalog #4061 – Cell Cycle Arrest siRNA (5 nmol)
  • Catalog #4062 – Apoptosis Inducing siRNA (5 nmol)
  • Catalog #4063 – Scrambled siRNA control, non-silencing siRNA (5 nmol)

Transfection Resource

Transfection Resource

Transfection optimization

The most vital aspect of a transfection is ensuring all conditions are optimized, including transfection reagent volume, oligo concentration, cell viability and passage number, lack of negative control activity, activity of positive control and calibrated incubator temperature, humidity and percentage of CO2.  Even if all these parameters are correctly addressed, transfection efficiency must be determined… Continue Reading

Importance of cell passage number and cell confluency for efficient transfection

The viability, confluency and passage number are all vital parameters for a successful transfection.  Here are helpful hints for each of these aspects: Viability Viability is the percentage of living cells in a suspension Determined using trypan blue exclusion Cells are considered to be healthy if viability is greater than 90% Confluency Confluency is the… Continue Reading

How much antibiotic required for stable cell selection?

Stable cell lines are a crucial laboratory tool that over-expresses a gene of interest in order to study gene functions, screen experimental drugs or produce therapeutic proteins (i.e. recombinant antibodies).  The cell lines will divide and continue to express the inserted transgene.  Briefly, exogenous plasmid DNA is transfected into a host cell line, which is… Continue Reading

Quantitate in vitro anti-proliferation experiments using a metabolic assay (Alamarblue, MTT) vs measuring protein abundance (Sulforhodamine B)

The need to measure cell proliferation effects is vast, including testing the effects of growth factors, novel pharmacological agents, cytotoxicity assessment or investigating cell activation.  Cell proliferation assays utilize a stain or substrate to make a correlation between the readout and the number of remaining cells post-treatment.  However, the means of assessing cell number varies… Continue Reading

What is the benefit of using an electroporation buffer

The process of electroporation exposes the cells to a high-voltage pulse of electricity to disrupt the phospholipid bilayer of the cell membrane causing the formation of temporary pores.  Any charged molecules (e.g. RNA, DNA) are forced into the cells thru the pores. Electroporation buffers are formulations that mimic cellular cytoplasm composition; thus, enhancing pore resealing… Continue Reading

Difference between technical and biological replicates

The basic definitions of technical and biological replicates are as follows: Technical replicates: a test performed on the same sample multiple times; i.e., if there are triplicate non-treated samples, a technical replicate would be testing sample #1 of the non-treated multiple times Biological replicates: a test performed on biologically distinct samples representing an identical time… Continue Reading

What is the best method to detach cultured cells?

Cultured adherent cells routinely need to be detached and collected for counting or passaging.  Detaching cells can be accomplished by either mechanical or enzymatic methods. Mechanical: cell scraping is a good option for cells that are sensitive to trypsin but can cause damage to cells; also can be used when collecting cellular components for western… Continue Reading

Forward Transfection or Reverse Transfection?

Forward and reverse transfection protocols each have their significant uses in research.  The main protocol difference between forward and reverse transfection is whether or not the cells are plated the day before transfection (as in forward transfection) or seeded at the same time of the transfection.  Forward transfection is commonly used in situations where the… Continue Reading

Stable transfection reagents and techniques

Stable transfection, sometimes called permanent transfection, is the integration of plasmid DNA into the chromosome of cancer cell’s DNA.  The creation of a stable cell line enables the researcher to analyze long term effects of the introduced gene.  Downstream studies incorporating the stable cell line includes overexpression of the gene insert for protein production, cell… Continue Reading

What transfection controls do I need to include in my experiment?

Scientists understand the importance of controls in experiment. There are at least three transfection controls that should be included on every transfection plate: a positive control, negative control and non-treated control. Positive Controls Transfecting a positive control ensures that the system being utilized is working and the delivery conditions are optimal.  The results of the… Continue Reading

What is the best antibiotic to use for stable cell selection in mammalian cells?

There is a long list of antibiotics available to researchers when applying selective pressure in the creation of a stable cell line. The choices include zeocin, hygromycin, blasticidin, puromycin and geneticin (G418). Researchers use different antibiotics due to cost or availability in their lab. However, in reference to using antibiotics for the creation of a… Continue Reading

How do I increase transfection efficiency?

Transfection is considered as a major laboratory method to integrate protein, RNA and DNA molecules into tissues and cells. Delivery of plasmid DNA molecules containing gene inserts, messenger RNA and small interfering RNA molecules that regulate gene expression (i.e. microRNA, siRNA) into the primary cells and cancer cell lines have been extensively utilized by scientists.… Continue Reading